Throughout this specification, various references are identified by author name in parentheses, The citation to the reference corresponding to the identified author can be found in the section entitled References Cited preceding the claims. The references in that section are hereby incorporated by reference,
Thaumatin and monellin are flavor active and flavor enhancing proteins. Thaumatin, as shown by thaumatin II, has a wide variety of uses as a flavor enhancing agent. The octapeptide known as xe2x80x9cdelicious peptidexe2x80x9d or xe2x80x9cbeefy meaty peptidexe2x80x9d (BMP) is reported to enhance flavor and produces an umami and sour taste, especially in beef. Monellin is recognized as a potently sweet protein. Recent work indicates that the 19 to 26 amino acid region of thaumatin is a possible active site for sweetness determination. (Slootstra et al. 1995). Substitution of this region with other amino acid sequences, particularly those having some homology with this region of thaumatin and believed to affect taste, may alter the properties of thaumatin to provide new and useful function.
Recent work with monellin indicates that the lysine located at position 4 in the A chain is a highly likely candidate for involvement as a component of the receptor interaction site of monellin. (Suami et al. 1996), Subsitution of this region with other amino acid sequences, particularly those having some homology with this region of monellin and believed to affect taste, may alter the properties of monellin to provide new and useful function.
The present invention provides new flavor active proteins. Specifically, the new proteins are modified versions of the thaumatin and monellin proteins. In thaumatin II amino acids in the region of 19 to 26 (amino acids 19-26 of SEQ. ID. NO.2), which appears to be a taste active region, are replaced with other amino acid sequences. Most important is the replacement of this region with the sequences for the octapeptide known as xe2x80x9cdelicious peptidexe2x80x9d or xe2x80x9cbeefy meaty peptide,xe2x80x9d i.e. LYS-GLY-ASP-GLU-GLU-SER-LEU-ALA (SEQ. ID. NO. 6), and the sequence that comprises the segment of protein from the fourth to the eleventh amino acid in the A chain of monellin, i.e. LYS-GLY-TYR-GLU-TYR-GLN-LEU-TYR (amino acids 4-11 of SEQ. ID. NO. 4; SEQ ID. NO.11). When four (4) of the 207 amino acids in the thaumatin b protein amino acid sequence were changed, a protein is produced that has a dramatic savory effect when evaluated with a complex salt enhancer such as L-arginine, ammonium chloride, tartaric acid, monopotassium glutamate, or ribotide. In the presence of the salt enhancer, the new protein with the BMP sequence has a beefy, meaty, brothy impression and mouth feel.
This invention further provides a modified monellin protein, either as A and B chains or as a single joined chain, wherein the amino acids in the region of four to eleven of the A chain (or the homologous section of single chain monellin), which appears to be the taste active region, are replaced with other amino acid sequences. Most important is the replacement of this region with the sequences for the octapeptide known as xe2x80x9cdelicious peptidexe2x80x9d or xe2x80x9cbeefy meaty peptide,xe2x80x9d i.e. LYS-GLY-ASP-GLU-GLU-SER-LEU-ALA (SEQ. ID. NO. 6), and the sequence that comprises the segment of protein from the amino acid region of 19 to 26 in thaumatin II, i.e. LYS-GLY-ASP-ALA-ALA-LEU-ASP-ALA (amino acids 19-26 of SEQ. ID. NO. 2; SEQ. ID NO.12).
This invention further provides the DNA sequences encoding the modified thaumatin and monellin proteins and for derivatives of such proteins and the DNA sequences of the derivatives. It also teaches methods for producing these proteins using genetic recombination techniques which can be used in a variety of microorganisms. According to the present invention, these proteins are expressable in yeast transformed by vectors comprising the nucleic acid sequences encoding the proteins. The microorganisms which have been transformed to express these proteins can be used to cultivate cell lines capable of producing the proteins on a large scale, Furthermore, this invention teaches the use of these proteins as flavor additives and enhancers in food.